The Novex® NuPAGE® SDS-PAGE gel system is a revolutionary high-performance polyacrylamide gel electrophoresis system that simulates the denaturing conditions of the traditional Laemmli system (Tris-Glycine SDS-PAGE gels). NuPAGE® SDS-PAGE gels  use a unique buffer formulation that maintains a low operating pH during electrophoresis.  This helps eliminate the “smiles” and poor resolution seen with Tris-Glycine SDS-PAGE gels.

The Novex® NuPAGE® SDS-PAGE gel system is designed for:

  • Superior protein band resolution and stability
  • Faster sample run times—35–50 minutes
  • Longer product shelf life—16 months from the date of manufacture
  • More efficient western blot transfer
  • Using gels from another company? Use the product selection guide to find our equivalent!

NuPAGE® Bis-Tris Pre-Cast gels

Ideal for separating small to mid-size MW proteins

Learn more about NuPAGE® Bis-Tris gels

 

NuPAGE® Tris-Acetate Pre-Cast gels

Ideal for separating large MW proteins

Learn more about NuPAGE® Tris-Acetate gels

NuPAGE® SDS-PAGE gel electrophoresis reagents

NuPAGE® SDS-PAGE gels are polyacrylamide gels made with Bis-Tris/HCl buffer (pH 6.4). During separation, the gels operate close to pH 7. Even though the NuPAGE® Bis-Tris gels do not contain SDS, they are formulated for denaturing gel electrophoresis applications only. In the Laemmli system, the gel is run at basic pH (~ pH 9.5); the residual unpolymerized acrylamide can react with cysteine and lysine residues of the proteins being separated. This may affect downstream analysis such as western blot transfer. At the neutral pH of NuPAGE® protein gels, these reactions take place several orders of magnitude more slowly than at the basic pH of Tris-Glycine gels, resulting in sharper bands and better resolution (Figure 1).
NuPAGE® Gel Comparison
Figure 1. Protein separation using (A) a Novex® NuPAGE® gel and (B) a traditional Tris-Glycine gel. The samples listed below were run on (A) a NuPAGE® 4–12% Bis-Tris gel in MES-SDS Running Buffer or (B) another manufacturer’s 4–20% Tris-Glycine gel. Note the high resolution of the sample bands in the NuPAGE®  protein gel. The poorly resolved “fuzzy” bands seen in the other manufacturer’s gel are a result of reoxidation of some disulfide bonds within the sample, leading to slight changes in migration rates. Lanes 1, 6, 7, 10: Mark12™ Unstained Protein Standard; lane 2: high protein load (4 proteins, 6 µg/protein); lane 3: Broad Range Standard; lanes 4, 5: E. coli extract; lane 8: SeeBlue® Pre-stained Protein Standard; lane 9: MultiMark Multi-colored Protein Standard.

NuPAGE® protein gels are available in two buffer systems: NuPAGE® Bis-Tris Gels and NuPAGE® Tris-Acetate Gels. The NuPAGE® Bis-Tris Gels provide superior separation of small to medium-sized proteins and employ a neutral-pH environment that minimizes protein modification. The NuPAGE® Tris-Acetate Gels are designed to provide optimal separation for large proteins at pH 8.1.

NuPAGE® Bis-Tris Gels are used in combination with either NuPAGE® MES or MOPS buffer. The NuPAGE® MES Buffer is recommended for resolving small proteins and for samples containing a broad range of protein sizes. The NuPAGE® MOPS Buffer is best for resolving medium-sized proteins.

NuPAGE® Tris-Acetate Gels are used with NuPAGE® Tris-Acetate SDS Running Buffer to resolve high molecular weight proteins (36–400 kDa) under denaturing conditions, or with Novex® Tris-Glycine Native Running Buffer to resolve high molecular weight proteins under non-denaturing (native) conditions.  Figure 2 shows the migration patterns of Sharp Protein Standards on NuPAGE® Bis-Tris Gels using MOPS and MES buffers, and NuPAGE® Tris-Acetate Gels using NuPAGE® Tris-Acetate SDS Running Buffer.

Migration patterns of protein standards on NuPAGE® Novex Gels

             

Figure 2. Migration patterns of Novex® Sharp Protein Standards (prestained and unstained) on NuPAGE® Bis-Tris Gels and NuPAGE® Tris-Acetate Gels. For optimal results, protein bands should migrate within the yellow shaded areas.

Sample preparation is a crucial step in successful protein analysis. The pH of the traditional Laemmli-style sample buffer changes from 6.8 to 5.2 when heated at 100°C.  This lower pH is known to induce aspartyl-prolyl (Asp-Pro) peptide bond cleavage, which leads to protein degradation. By maintaining a >7.0 pH environment, the NuPAGE® LDS Sample Buffer preserves protein integrity by minimizing this Asp-Pro cleavage. Figure 3 demonstrates that sample integrity is maintained throughout electrophoresis with the NuPAGE® system; protein degradation is seen in samples prepared with Laemmli (Tris-glycine) sample buffer.  The NuPAGE® Antioxidant running buffer additive greatly minimizes protein oxidation during electrophoresis and keeps reduced protein bands sharp and clear.  

Novex® NuPAGE® SDS-PAGE Gel System
    

Figure 3. Integrity of samples is maintained throughout electrophoresis with the Novex® NuPAGE® SDS-PAGE Gel System (left), compared to samples prepared with Laemmli (Tris-glycine) sample buffer (right).

Increased Gel Stability

The NuPAGE® protein gels are discontinuous SDS-PAGE gel systems that operate in the same way as the traditional Tris-glycine system but are prepared at a lower pH. This neutral-pH environment allows a much longer shelf life (1 year for NuPAGE® Bis-Tris Gels and 8 months for NuPAGE® Tris-Acetate Gels).

Efficient Western Blot Transfer

The NuPAGE® Transfer Buffer maintains neutral pH and prevents reoxidation of reduced samples during protein transfer to a membrane. This avoids sample modifications that can occur at the alkaline pH of traditional transfer buffers and maintains sample antigenicity. NuPAGE® Bis-Tris Gels are able to separate proteins using lower acrylamide concentrations than are required for Tris-glycine gels. This more open gel matrix allows for more efficient transfer of proteins to membranes during western blotting.

Best All-Around Performance

For routine protein separation, use the NuPAGE® SDS-PAGE Gel System for superior performance. In addition, the high-resolution separation and reduced protein modification and degradation properties make this system suitable for even the most demanding applications. Because the same protein separation can be achieved at lower acrylamide concentrations using NuPAGE® Bis-Tris Gels compared to Tris-glycine gels, western blot transfers are also more efficient.

For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.